Abstract
Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).
Highlights
Cryogenic electron microscopy image collection and analysis technique referred to as single-particle analysis (SPA) allows near-atomic structural resolution of purified biomolecular complexes
Subtomograms suffer from the so-called missing wedge artifacts, which are due to inability to include in the 3D reconstruction the images from all orientations
This article presents HEMNMA-3D, the first cryogenic electron tomography (cryo-ET) subtomogram data analysis approach to study continuous conformational variability of biomolecular complexes, which maps a set of subtomograms into a space of conformations using a reference model and its normal modes
Summary
Cryogenic electron microscopy (cryo-EM) image collection and analysis technique referred to as single-particle analysis (SPA) allows near-atomic structural resolution of purified biomolecular complexes (in vitro). It is based on the principle of reconstructing a three dimensional (3D) structure from two dimensional (2D) parallel-beam projection images of vitrified specimens containing many copies of the same macromolecular complex at unknown orientations and positions. A tomographic reconstruction typically contains hundreds of copies of a target biomolecular complex at unknown orientations and positions These copies are identified and extracted into individual (single-particle) volumes called subtomograms, either manually or semi-automatically (via template matching methods). Due to the low SNR and the missing wedge artifacts, cryo-ET data processing is mainly based on rigid-body aligning and averaging many subtomograms to enhance the data quality and reveal the targeted biomolecular structure (Leigh et al, 2019)
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